The basis of the studies proposed in this project reflect the findings emanating from the initial and longstanding observations in our lab that regulatory T cells are activated following interactions with normal intestinal epithelial cells. These studies also documented defects in CD8+ Treg activation when epithelia cells derived from IBD patients were used to activate T cells. During the course of defining the repertoire of regulatory T cells present in normal and inflamed intestine during the last granting cycle, we identified a novel population of CD4+ FoxP3/IL17 double positive cells in the lamina propria of CD but not UC or normal patients. These cells share phenotypic characteristics of Th17 cells with secretion of IL17, IL22. IL21 while expressing high levels of CCR6, CD161, and RORyt. However, unlike conventional Th17 (cells and similar to FoxP3+ Tregs, they express high levels of CD101 and low levels of CD127, exhibit a similar TcR repertoire (BV usage) and are functionally suppressive in in vitro co-culture systems. FoxP3+IL-17 producing cells are imprinted for gut homing, as indicated by high levels of CCR6, CD103 and the integrin a4B7 expression. These cells secrete IFNy but not IL10 or TGFB. Thus they represent a novel cell population that could provide useful insights into lineage commitment of Tregs versus Th17 cells. We propose that these cells sit at the crossroads between Treg and Th17 cells and that further commitment to either lineage results from microenvironmental cues present in the tissues. The current proposal seeks to characterize these cells and define factors involved in their activation. Most importantly we aim to detemiine the microenvironmental cues that allow these cell to commit to a Th17 (more likely in CD) rather than a regulatory lineage. We will: 1) Define the functional and phenotypic properties of the CD4+ FoxP3/IL17 double positive cells derived from CD; 2) Define the microenvironmental cues leading to a transcriptional program that regulates lineage commitment of these cells. In collaboration with Dr. Xiong in Project 3 (IRF8), assess the interactions between FoxP3 and RORyt and determine factors that allow for negative regulation of both transcription factors. 3) Identify counterparts of these cells in murine models of ileitis/colitis or murine infection models in collaboration with Drs. Lira and Blander to establish the conditions required for their generation in vivo.